A. Field of the Invention
The present invention relates to a method for DNA comparative analysis in a plurality of samples and a sample preparation method for the DNA analysis.
B. Description of the Prior Art
With the progress of genome analysis, the first stage of the genome project, where the analysis of genome structures by DNA sequencing is the major subject, is going to the end and the genome analysis comes to the second stage of understanding gene functions. The genetic information in genome sequences has to be translated to a protein through mRNA. The genes expressed in a cell at some moment can be determined by detecting mRNAs in the cell. Genetic characteristics of individuals are dependent on various differences in their genome sequences. The analysis of mRNAs in cells or tissues and the comparative analysis of DNA sequences for individual genes are necessary for understanding the gene functions. Especially the analysis of species and amounts of mRNAs in cells is important to know what is going on in the cells. Usually, cDNA (complementary DNA), which is produced by complementary strand extension reactions with a DNA polymerase and a primer hybridizing to each mRNA, is used for the analysis instead of mRNA because mRNAs are easily decomposed by RNase that is in cells.
The scanning of all the cDNAs (or mRNAs) in cells or tissues is called as gene expression profiling. As the size of each cDNA is usually very long to be sequenced or to be analyzed by gel electrophoresis, a part of the sequence is selected as the signature sequence of the cDNA to be analyzed. Each of the signature sequences of cDNAs is amplified and analyzed by gel electrophoresis or by hybridization with a DNA probe array. At first, the signature regions of cDNAs are amplified by PCR (Polymerase Chain Reaction) and then the relative abundance of each signature fragment is analyzed. The key point of the method is how to amplify each of signature regions without losing the relative abundance information. The relative abundance information is frequently lost during the PCR process because the amplification factors of each PCR reaction are dependent on the precise conditions and the sequence of the target cDNA fragment. The PCR amplification for plural of target DNA fragments should be carried out simultaneously to keep the amplification conditions the same. However, it is not so easy because the primers used for amplifying the target DNA fragments frequently interact with each other to produce undesired new DNA fragments which disturb the accuracy and reliability of the gene expression profiling.
The present invention relates to a method for carrying out the simultaneous PCR amplification of various cDNA fragments for quantitative cDNA analysis such as gene expression profiling. The invention also relates to the method for recovering PCR products and the sample preparation for DNA diagnostics. In PCR amplification, two primers are designed to hybridize on the template DNA at predetermined positions. The base sequence of the template DNA sandwiched with the two primers is amplified by repetitive complementary strand extension reactions with the primers. The number of copies of the target DNA fragments increases by several orders of magnitude by PCR. In the case of gene expression profiling, a DNA sample contains various cDNAs fragment species. Many should be analyzed quantitatively. The method requires the PCR amplification of plural of target cDNA fragment species simultaneously. When the PCR amplification of a plurality of DNA fragments or sequences is carried out, artificial fragments are frequently produced through unexpected reactions among primers and DNAs. However, the isolation of the amplified components is labor intensive. Consequently, only one pair of primers is used at a time for PCR amplification. When many target DNA fragment species have to be analyzed, many PCR reactions are required. This is very labor intensive.
On the other hand, the comparative analysis for two or more kinds of DNA fragments is an important subject and is extensively investigated. However, since the amplification rate in PCR depends greatly on the reaction conditions, the comparison of groups of DNA fragments which are obtained under different PCR conditions, namely, groups of DNA fragments which are independently obtained by amplification, has been disadvantageous in that it prohibits quantitative investigation. Factors capable of affecting PCR include the reaction temperature, the base sequences of primers, the amounts of reagents, the kinds and amounts of contaminants, etc. It is considerably difficult to make these factors the same for different reactions.
A PCR technique for quantitative and comparative analysis for one DNA fragment species in various samples such as tissues has recently been developed. This method is called adaptor-tagged competitive PCR (ATAC PCR). Now the target of the analysis is the same DNA fragment species in different DNA samples (for example, different sample numbers are used to identify those samples; sample number 1-sample number 9). There are plural of samples containing various DNA species to be compared. The method can carry out comparative analysis of DNA fragment species belonging to different samples by putting tags depending on the samples. The tagging is taken place by changing the lengths of oligomers connected to the DNA fragments as follows. An oligonucleotide having a known base sequence is connected to each end of the DNA fragment species. The known base sequence is composed of a common base sequence for the hybridization of a primer and a tagging base sequence for discriminating the plurality of the samples containing various DNA species. To separate DNA fragment species produced from different samples, the tagging sequences are designed so as that their lengths are different from sample to sample. In ATAC PCR analysis, only one target DNA fragment species in various samples is analyzed at a time. Each sample contains the target DNA fragment sequences at different ratios. The priming site for PCR amplification is also the same for different DNA fragments. The only difference in the targets is the lengths of the tagging sequence region. Consequently, all the target DNA fragments can be amplified at the same amplification rate while the tagging sequences are kept tagged through the amplification. At least one of the primers used in PCR amplification is labeled with fluorophore. The fluorophore labeled DNA fragment amplified by PCR are analyzed by gel electrophoresis coupled with fluorescence detection. The DNA fragments originated from different DNA samples appear in the different positions in an electropherogram which is used for the comparative analysis of the gene expression.